Process for producing enriched fractions of tetrahydroxycurcumin and tetrahydrotetrahydroxy-curcumin from the extracts of Curcuma longa

ABSTRACT

A process for producing an enriched fraction of tetrahydoxycurcumin containing, tetrahydroxycurcumin, demethylcurcumin, demethylmonodemethoxycurcumin and bisd.emethoxycurcumin and colorless tetrahydroderivatives thereof. The process consists of demethylation of natural curcumins, obtained, in turn, from the organic solvent extract of turmeric from  Curcuma  species. The said enriched fraction of tetrahydroxycurcumin is subjected to hydrogenation to get colorless tetrahydrotetrahydroxycurcumin enriched fraction. The enriched fractions of tetrahydroxycurcumin and colorless tetrahydrotetrahydroxycurcumin exhibits potent antioxidative action and reduces inflammation.

The invention relates to a process for producing enriched fractions oftetrahydroxycurcumin and its tetrahydroderivative,tetrahydrotetrahydroxycurcumin. These enriched fractions exhibit potentantioxidative action and reduce inflammation. The products of thepresent invention are suitable as food additives, nutraceutical orcosmoceutical applications.

TECHNICAL FIELD

Free radicals play a major role in the initiation and progression of awide range of pathological diseases like cancer, Alzheimer's,Parkinson's, and cardiovascular diseases. In the food industry, freeradicals have been found to be responsible in the deterioration of foodsduring processing and storage In view of this, considerable attentionhas been given to the addition of antioxidants in foods andsupplementation of antioxidants to biological systems to scavenge freeradicals. The antioxidative compounds can be classified into two types:phenolics and β-diketones. Phenolic compounds exert their antioxidantactivity by acting primarily as hydrogen atom donators, therbyinhibiting the propagation of radical chain reactions. Antioxidantpotential of the phenolics depends on the number and arrangement ofphenolic hydroxyl groups, as well as the nature of the othersubstituents on the aromatic rings. Few natural products, likecurcuminoids have both phenolic and β-diketone groups in the samemolecule and thus became strong antioxidants. Curcumins, the phenolicdiarylheptanoids, are characteristic yellow coloured constituents ofturmeric (Curcuma longa) and are widely used in neutaceuticals, foodsand cosmetics. FIG. 1, shows the chemical structures of curcumins:curcumin, monodemethoxycurcumin, bisdemethoxycurcumin,tetrahydroxycurcumin and herein after referred as C, MDC, BDC, TC. Thesecompounds were reported to poseses antioxidant, anti-inflammatory,anticancer, Alzheimer's and antiviral properties. Of all the fourcurcumins, TC shows most potent anticancer, antioxidative andinflammatory activity. But the natural curcumin mixture contains TC invery low concentration (0-5%) depending on the raw material.

Presently, there has been a tremendous surge in demand fornon-steroidal, plant based anti-inflammatory agents. 5-Lipoxygenase isthe key enzyme for the biosynthesis of leukotrienes and 5(S)-HETE, theimportant mediators for inflammatory, allergic and obstructive process,from arachidonic acid. 5-Lipoxygenase is the target enzyme foridentifying inhibitors, which have potential to cope with a variety ofinflammations and hypersensitivity-based human diseases includingasthma, arthritis, bowl diseases such as ulcerative colitis andcirculatory disorders such as shock and ischaemia.

Because of proven safe, non-toxic nature of curcuminoids and the lack ofenriched tetrahydroxycurcumin fraction to address the above problems, itis therefore an object of the present invention to provide enrichedtetrahydroxycurcumin as a safe dietary supplement, which treatsinflammatory diseases, free radical mediated diseases and nutraceuticaland cosmetic applications.

DISCLOSURE OF THE INVENTION

The present invention relates to a process for manufacturing an enrichedfraction of tetrahydroxycurcumin from the extract of turmeric (Curcumaspecies), which contains tetrahydroxycurcumin (TC), along withdemethylcurcumin (DC), demethyldemethoxy-curcumin (DMDC) andbisdemethoxycurcumin (BDC) as minor compounds. The present inventionalso encompasses the above said fraction for administration to animalsincluding mammals, and a method of treating various inflammatoryconditions and also a method of preventing various oxidative diseaseconditions by administering the inventive fraction.

The other objective of the present invention is a process for producingan enriched colorless tetrahydrotetrahydroxycurcumin (THTC) from theabove said enriched tetrahydroxycurcumin fraction and a method fortreating various inflammatory conditions and also for preventing variousoxidative disease conditions by administering the inventive colorlesstetrahydrotetrahydroxycurcumin fraction.

Yet another object of the present invention is a method for isolatingTC, DC, DMDC and BDC in pure form by column chromatography, followed bycrystallization.

DETAILED DISCLOUSURE OF THE INVENTION

Organic solvent extracts of the curcuma species, particularly Curcumalonga have been found to contain a total of four curcumins. These areshown in FIG. 1 and are represented by C, MDC, BDC and TC. Concentrationof tetrahydroxycurcumin, designated as TC in the FIG. 1, amounts only inthe range of 0.1 to 5% in the natural curcumin fraction (Mimura Akio etal., U.S. Pat. No. 5,266,344, 1993). Among the four curcumins, TC showedmost potent anticancer, antioxidant and anti-inflammatory activities.However, there is no report on the enrichment process of TC in curcuminmixture.

The present invention is aimed at enriching the concentration of TC inthe curcumin fraction to a desired concentration upto 100%. Anotherobjective of this invention is to convert less potent curcumins presentin the extract by demethylating them to highly potent antioxidativedemethylcurcumins. The said demethylation leads to a fraction, whichcontains higher concentration of TC. Pure TC could also be obtained fromthe TC enriched fraction by a simple purification process.

A combination of simple chemical reaction and purification bychromatographic method achieves these objectives.

The present inventive enriched tetrahydroxycurcumin fraction obtainedfrom the natural Curcuma extract contains a total of four compounds.These are shown in FIG. 2 and are represented by TC, DC, BMDC and BDC.Concentration of TC is in the range of 10-100%.

The process involves demethylation of Curcuma longa extracts by Lewisacid catalyst in a suitable solvent to an enriched fraction oftetrahydroxycurcumin. An organic base and a catalyst in addition toaluminum halides are used for demethylation. Dry material obtained aftersimple work-up showed 50-80% of TC by HPLC analysis.

The Lewis acid catalysts such as aluminum chloride, aluminum bromide,aluminum iodide, broron tribromide or boron trichloride-methyl sulfidecomplex or sodium salt of N-methylaniline or sodium ethanethiolate orlithium chloride in dimethyl formamide or beryllium chloride are used.Solvents such as chloroform, dichloromethane, dichloroethane and ethylacetate or mixtures thereof are used. Organic bases such as pyridine,triethylamine, piperidine are used and catalyst is selected from sodiumiodide or potassium iodide or PTC catalyst such as tetrabutylammoniumbromide etc. are used.

Pure TC is obtained from the enriched tetrahydroxycurcumin fraction bychromatographic methods. Solid supports such as silica gel, reversedphase silica, alumina and sephadex can be used in the process.Chromatographic techniques are selected from gravity column, flashchromatography, reversed phase chromatography, preparative high pressureliquid chromatography and the combinations thereof. Solvents such asacetone, chloroform, dichloromethane, ethyl acetate, hexane and watereither alone or in combination to run a gravity column or flash columnor medium pressure column are used.

This invention relates to a process for producing 50% to 100% of TC fromthe extracts of Curcuma species particularly Curcuma longa, whichcomprises the steps of demethylation of the said extracts, followed bychromatographic separation to obtain a fraction enriched in TC in therange of 50 to 100%.

The invention also relates to a process for isolating all the fourcompounds in the present inventive tetrahydroxycurcumin fraction in pureform by column chromatography using polar and non-polar solvents aseluents, followed by crystallizations. The structures of the isolatedpure TC, DC, DMDC and BDC (FIG. 2) have been confirmed by their physicaland spectral data (IR, NMR and mass).

Though the above enriched tetrahydroxycurcumin fraction exhibits strongantioxidative activity than the curcumins, but its application may belimited because of its strong yellow color. For application in colorlessfoods and cosmetics, we have invented a colorlesstetrahydrotetrahydroxycurcumin fraction by a process of hydrogenation.The process of hydrogenation of curcumins could also occur naturally inthe gastrointestinal tract. The tetrahydrotetrahydroxycurcumin fractionis also strong antioxidant similar to tetrahydroxycurcumin combined withthe lack of yellow color render them useful in achromatic food andcosmetic applications that currently employ conventional syntheticantioxidants.

So the invention is also aimed to obtain a colorlesstetrahydrotetrahydroxycurcumin (THTC) enriched fraction.

The presently invented colorless tetrahydrotetrahydroxycurcumin enrichedfraction from the said tetrahydroxycurcumin fraction has been found tocontain a total of four tetrahydro compounds. These are shown in FIG. 3and are represented by tetrahydrotetrahydroxycurcumin (THTC),tetrahydrodemethylcurcumin (THDC),tetrahydrodemethylmonodemethoxycurcumin (THBMDC) andtetrahydrobisdemethoxycurcumin (THBDC). Concentration of THTC is in therange of 10-100%.

The process involves the hydrogenation of TC enriched fraction byreducing the double bonds using metal catalyst in a suitable solvent anda hydrogen gas or hydrogen donor to an enriched fraction oftetrahydrotetrahydroxycurcumin (THTC). An organic base is also used ifdesired.

The metal catalysts such as palladium-carbon, Raney-nickel, platinum,zinc or manganese are used. Solvents such as ethyl acetate, acetone,methanol, ethanol, isopropanol, tetrahydrofuran, dioxane or mixturesthereof are used. Hydrogen donors such as formic acid, acetic acid,propanoic acid or ammonium formate are used. Organic bases such astriethyl amine, trimethyl amine or piperidine are used.

Pure THTC is obtained from the enriched fraction oftetrahydrotetrahydroxycurcumin by chromatographic methods as describedabove. Pure THTC could also be obtained by the hydrogenation of pure TC.

The invention also describes a method of treating inflammatoryconditions by use of the above said enriched tetrahydroxycurcumin orpurified TC or colorless tetrahydotetrahydroxycurcumin and the activitywas supported by the measurement of 5-lipoxygenase activity. From thepercentage of 5-lipoxygenasc inhibitory values (Table 1) of the presentinventive tetrahydoxycurcumin fraction or pure TC ortetrahydrotetrahydroxycurcumin showed potent 5-lipoxygenase activity andthe activity is superior to that of existing commercial curcumin mixtureand comparable to that of AKBA, a potent 5-lipoxygenase inhibitor fromBoswellia serrata.

The invention also describes a method of treating or preventing radicalmediated complications in humans or in foods by the use of above saidenriched fraction of tetrahydroxycurcumin or purified TC or colorlesstetrahydrotetrahydroxycurcumin and the activity was supported by themeasurement of superoxide and DPPH radical scavenging activity. From thepercentage of inhibitory values (Table 2) of the present inventiveenriched tetrahydroxycurcumin fraction or pure TC or colorlesstetrahydrotetrahydroxycurcumin showed potent antioxidative activity andthe activity is superior to that of existing commercial curcuminmixture, BHT (butylatedhydroxytoluene), BHA (butylated hydroxyanisole),vitamin C and vitamin E.

The invention also describes use of enriched tetrahydroxycurcuminfraction containing 70-100% of TC for treating anti-inflammatoryconditions. The anti-inflammatory activity was demonstrated by thecarragenean induced paw edema method. The above enriched TC fractionshowed 20.56% inhibition at 50 mg concentration, whereas the standarddrug, diclofenac sodium showed 63.10% inhibition at 25 mg concentration.From these results it is clear that the present inventivetetrahydroxycurcumin fraction showed significant anti-inflammatoryactivity.

A further aspect of the present invention is a pharmaceuticalformulation comprising a enriched fraction of tetrahydroxycurcuminfraction or TC or colorless tetrahydrotetrahydroxycurcumin as describedabove in a pharmaceutically acceptable carrier (e.g., an aqueous or anon aqueous carrier).

A still further aspect of the present invention is a method of treatinginflammatory diseases, comprising administering to a human or animalsubject in need thereof a therapeutically effective amount (e.g., anamount effective to treat, slow the progression of, etc.) of a enrichedtetrahydroxycurcumin fraction or pure TC or colorlesstetrahydrotetrahydroxycurcumin as described above.

A still further aspect of the present invention is a method ofpreventing radical mediated diseases, comprising administering to ahuman or animal subject in need thereof a therapeutically effectiveamount (e.g., an amount effective to treat, slow the progression of,etc.) of a enriched tetrahydroxycurcumin or pure TC or colorlesstetrahydrotetrahydroxycurcumin as described above.

The invention is described in the examples given below which areprovided by a way of illustrations only and should not be construed tolimit the scope of the present invention.

EXAMPLE 1

Enriched fraction of tetrahydroxycurcumin To an ice cold solution ofcurcumin mixture (95%, 55 g) in EtOAc (2.5 L), was added aluminumchloride (150 g) followed by the dropwise addition of pyridine (350 mL)for 15 min. and the reaction mixture was heated under reflux for 7 h.After cooling the reaction mixture to 10° C., cold dil. HCl (20%) wasadded to decompose aluminum chloride complex and extracted with ethylacetate (5×1.0 L). The combined ethyl acetate layer was washed withwater, brine and dried over anhydrous sodium sulfate. The solvent wasfiltered and evaporated the solvent. The residue was diluted withchloroform (100 mL) and kept for 10 h and the solid was filtered anddried to give the product (21 g, 38%).

HPLC analysis:

TC = 78.40 DC = 4.11 DMDC = 11.52 BDC = 0.86 Total = 94.89%

EXAMPLE 2

Enriched fraction of tetrahydroxycurcumin. To an ice cold solution ofcurcumin mixture (95%, 110 g) in EDC (4 L), was added dry aluminumchloride (160 g) followed by the dropwise addition of pyridine(distilled, 200 mL) for 15 min. followed by sodium iodide (5 g) and thereaction mixture was heated under reflux for 27 h. After cooling thereaction mixture to 10° C., diluted with water (2 L), acidified with HCl(50%) and stirred for 15 min. The organic layer was separated and waterwas added to the aqueous layer until 10 L volume. The aqueous layer wasstirred at rt for 2 h and settled for 16 h. The solids formed werefiltered and washed with water (2.5 L) and dried to give the crudemixture of demethylcurcumins, 94 g, which was stirred in ethyl acetate(2.5 L) at 70-80° C. for 1 h, filtered through supercel and evaporatedthe solvent to give the product, 84 g. This solid was stirred withdiethyl ether (500 mL) at rt for 30 min. and filtered, dried to give theproduct 58 g.

HPLC analysis:

TC = 75.68 DC = 6.32 DMDC = 11.24 BDC = 1.1 Total = 95.31%

EXAMPLE 3

Isolation of pure TC[1,7-Bis(3,4-dihydroxyphenyl)-1,6-heptadiene-3,5-dione]. Thedemethylcurcumin mixture (1 Kg, 75% TC) from example 2, was adsorbedover silica gel (100-200 mesh, 2 Kg) and chromatographed over silica gelcolumn using chloroform-methanol (95:5) as eluents to give pure TC,which was crystallized from chloroform-methanol as yellow color powder(0.5 Kg), mp 302-304° C.; IR (KBr): 3488, 3386, 1629, 1617, 1600, 1271,1289, 1142, 1120, 955 cm⁻¹; ¹H NMR (DMSO-d₆) δ 6.06 (1H, s, H-4), 6.56(2H, d, J=15.6 Hz, H-2,6), 6.77 (2H, d, J=8.3 Hz, H-5′,5″), 7.00 (2H, d,J=1.8 Hz, H-2′,2″), 7.06 (2H, dd, J=8.3, 1.8 Hz, H-6′,6″), 7.44 (2H, d,J=15.6 Hz, H-1,7), ¹³C NMR (DMSO-d₆) δ 183.1, 147.8, 145.1, 140.8,127.7, 126.5, 121.9, 115.9, 114.5, 100.9; LC-MS m/z (%): (ESI-negativemode) 339 [(M−H)⁻, 100].

EXAMPLE 4

Isolation and charactarisation of other ingradients intetrahydroxycnrcumin fraction. The demethylcurcumin mixture (1 Kg) fromexample 2, was adsorbed over silica gel (100-200 mesh, 2 Kg) andchromatographed over silica gel column using chloroform-methanol (95:5)as eluents to give pure DC, DMDC and BDC. The following are the spectraldata of the isolated compounds.

DC[1-(3,4-dihydroxyphenyl)-7-(3-methoxy-4-hydroxyphenyl)-1,6-heptadiene-3,5-dione].Yellow color powder, mp 164-166° C.; IR (KBr): 3484, 1621, 1267, 1132,1140, 964 cm⁻, ¹H NMR. (DMSO-d₆) δ 3.82 (3H, s, Ar—OCH₃), 6.04 (1H, s,H-4), 6.53 (1H, d, J=16.0 Hz, 11-2 or H-6), 6.74 (1H, d, J=16.0 Hz, 11-2or H-6), 6.76 (1H, d, J=8.5 Hz, H-5′), 6.80 (1H, d, J=8.3 Hz, H-5″),7.07 (1H, dd, J=8.5, 1.8 Hz, H-6′), 7.00 (1H, d, J=1.8 Hz, H-2′), 7.12(1H, d, J=1.8 Hz, H-2″), 7.29 (1H, dd, J=8.3, 1.8 Hz, H-6″), 7.44 (1H,d, J=16.0 Hz, H-1 or H-7), 7.51 (1H, d, J=16.0 Hz, H-1 or H-7); ¹³C NMR(DMSO-d₆): 183.0, 183.2, 148.6, 147.9, 147.7, 145.1, 140.8, 140.7,126.5, 122.8, 121.9, 121.0, 120.7, 115.9, 115.6, 114.6, 111.0, 101.0,55.4; EIMS m/z (%): 354 (M⁺, 16), 336 (20), 328 (54), 271 (71), 192(53), 191 (30), 177 (100), 167 (47), 163 (49), 150 (40), 149 (24), 145(84), 135 (48), 117 (42), 89 (57), 77 (43).

DMDC[1-(4-hydroxyphenyl)-7-(3,4-dihydroxyphenyl)-1,6-heptadiene-3,5-dione].Yellow color powder, mp 218-220° C.; IR. (KBr): 3338, 1627, 962 cm⁻¹; ¹HNMR (DMSO-d₆) δ 6.06 (1H, s, H-4), 6.59 (1H, d, J=15.8 Hz, H-2 or H-6),6.69 (1H, d, J=15.8 Hz, H-2 or H-6), 6.83 (1H, d, J=8.2 Hz, H-5″), 6.79(2H, d, J=8.0 Hz, H-3′,5′), 7.03 (1H, s, H-2″), 7.09 (1H, d, J=8.2 Hz,H-6″), 7.45 (1H, d, J=15.9 Hz, H-1 or H-7), 7.47 (1H, d, J=15.9 Hz, H-1or H-7), 7.57 (2H, d, J=8.0 Hz, H-2′,6′), 9.17 (1H, br s, Ar—OH), 9.63(1H, br s, Ar—OH), 10.04 (1H, br s, Ar—OH); EIMS m/z (%): 324 (M⁺, 18),306 (8), 299 (34), 298 (90), 242 (30), 241 (100), 163 (49), 161 (26),162 (38), 147 (87), 110 (43), 119 (39), 91 (21), 44 (34).

BDC [1,7-bis(4-hydroxyphenyl)-1,6-heptadiene-3,5-dione]. Yellow colorpowder, mp 222-224° C.; IR (KBr): 3211, 1620, 1600, 1269, 1168, 1140,955, 831 cm⁻¹; ¹H NMR (DMSO-d₆) δ 6.03 (1H, s, H-4), 6.68 (2H, d, J=16.0Hz, H-2,6), 6.80 (4H, d, J=8.0 Hz, H-3′,5′,3″,5″), 7.50 (2H, d, J=16.0Hz, H-1,7), 7.55 (4H, d, J=8.0 Hz, H-2′,6′,2″,6″); EIMS m/z (%): 308(M⁺, 20), 290 (14), 159 (36), 146 (100), 147 (87), 119 (38), 106 (42),90 (42), 65 (32).

EXAMPLE 5

Enriched fraction of tetrahydrotetrahydroxycurcumin. To a solution ofenriched fraction of tetrahydroxycurcurnin (95%, 25 g) from example 2,in ethyl acetate (100 mL) was added triethyl amine (50 mL) andpalladium-calcium carbonate (5%, 3.75 g) followed by dropwise additionof formic acid (8 mL) for 1 h at refluxing temperature. The reactionmixture was refluxed for 8 h. Formic acid (4.5 mL) was addedperiodically at 2 h intervals. After completion of the reaction,solvents were distilled off (appr. 50 mL). The cooled reaction mixturewas acidified with HCl (50%) and diluted with ethyl acetate (100 mL).The solution was filtered through supercel and separated the ethylacetate layer. The aqueous layer was further extracted with ethylacetate (2×100 mL) and the combined ethyl acetate layer was washed withwater, brine and dried over sodium sulfate. The solution was filteredand evaporated upto 10 mL volume and diluted with hexane (20 mL). Thesolution was passed through silica gel column using chloroform-methanol(10%, 100 mL) as eluents to give the product (13 g) as a low meltingsolid.

HPLC Analysis:

THTC = 72.86% THDC = 15.98% THDMDC = 7.56% THBDC = 0.12% Total = 96.39%

Antioxidant Activity

(a) Superoxide free radical scavenging activity. The superoxide freeradical scavenging activity was determined by the NBT (nitro bluetetrazolium) method. The reaction mixture contained EDTA (6.6 mM), NaCN(3 pg), riboflavin (2 NBT (50 μM), various concentrations of the testdrug in ethanol and a phosphate buffer (58 mM, pH 7.8) in a final volumeof 3 ml. Optical density was measured at 560 nm. The test tubes wereuniformly illuminated with an incandescent lamp for 15 min, after whichthe optical density was measured again at 560 nm. The percentageinhibition and superoxide radical generation was measured by comparingthe absorbance values of the control and those of the test compounds.IC₅₀ values were obtained from plot of the concentration in μg againstthe percentage inhibition.

(b) DPPH free radical scavenging activity. DPPH(1,1-diphenyl-2-picryl-hydrazyl) radical scavenging activity wasmeasured based on the reduction of methanolic solution of the coloredDPPH. Free radical scavenging ability of the test drug in ethanol addedto the methanolic solution of DPPH is inversely proportional to thedifference in initial and final absorption of DPPH solution at 516 nm.The reaction mixture contained 1×10⁻⁴ mM methanolic solution of DPPH andvarious concentrations of test drugs. The percentage inhibition wasdetermined by comparing the absorbance values of test and control tubes.

5-Lipoxygenase activity: The enriched fraction of tetrahydroxycurcuminmixture, pure TC and tetrahydrotetrahydroxycurcumin fraction werescreened for their 5-Lipoxygenase inhibitory potential usingcolorimetric method. The assay mixture contained 50 mM phosphate bufferpH 6.3, 5-Lipoxygenase, various concentrations of test substances indimethyl sulphoxide and linoleic acid in a total volume of 0.5 mL, after5 min incubation of above reaction mixture, 0.5 mL ferric xylenol orangereagent was added and OD was measured after two minutes at 585 nm usingspectrophotometer. Controls were run along with test in a similar mannerexcept using vehicle instead of test substance solution. Percentinhibition was calculated by comparing absorbance of test solution withthat of control.

Anti-inflammatory activity (Carragenin induced paw edema method): Priorto the experiment all the animals (Albino wistar rats of either sexweighing between 180-300 g) fasted at ad libitum water and were weighed,numbered and randomly divided into groups, each containing 3 animals.Initial paw volumes were measured using plethesmometer and noted. Allthe groups were treated with corresponding test substance orally usinggastric tube. Control group was treated with 10 mL/Kg vehicle (0.5%,carboxymethyl cellulose sodium salt). After 30 minutes, all the animalswere injected subcutaneously at subplantar region of left hind paw 1%carrageenin 0.1 mL using hypodermic needle. All the animals wereadministered water 20 mL/Kg body weight and kept devoid of water for 3 h(maintained uniform hydration). After 3 h, paw volumes of all theanimals were measured twice and average volume from two measurementswere recorded. The % of inhibition of paw edema was calculated bycomparing paw edema of test substance treated groups with that ofcontrol groups.

TABLE 1 Antioxidant activity Superoxide (NBT) DPPH S. No. Name of thecompound IC₅₀ in μg IC₅₀ in μg 1 Natural curcumins mixture 27.5 2.9(95%) 2 Enriched tetrahydroxycurcumin 3.1 1.1 fraction (95%) 3 Puretetrahydroxycurcumin 2.5 1.1 (TC) 4 Tetrahydrocurcumins mixture >100 2.95 Enriched 3.0 1.5 Tetrahydrotetrahydroxycurcumin fraction 6 Pure 4.01.6 tetrahydrotetrahydroxycurcumin (THTC) 7 BHT 90 5.3 8 BHA 174 6.1 9Vitamin C 150 4.4 BHA: Butylated hydroxyanisole; BHT: Butylatedhydroxytoluene The lower the IC₅₀ value, the higher is the antioxidantactivity.

TABLE 2 5-Lipoxygenase inhibitory activity Concentration % of S. No.Name of the compound in μg/mL inhibition 1 Natural curcumins mixture 16022 (95%) 2 Enriched tetrahydoxycurcumin 4 51 fraction (95%) 3 Puretetrahydroxycurcumin (TC) 16 60 4 Tetrahydrocurcumins mixture 100 Nil 5Enriched 50 46 tetrahydrotetrahydroxycurcumin fraction 6 Pure 80 0.2tetrahydrotetrahydroxycurcumin (THTC) 7 AKBA 41 55 8 NDGA 24 60 AKBA:Acetyl-keto-boswellic acid; NDGA: Nordihydroguaiaretic acid

The invention claimed is:
 1. A composition comprisingtetrahydrotetrahydroxycurcumin in the range of 30-80%;tetrahydrodemethylcurcumin in the range of 4-20%;tetrahydrodemethylmonodemethoxycurcumin in the range of 5-25% andtetrahydrobisdemethoxycurcumin in the range of 0.1-10%.
 2. Apharmaceutical or dietary supplement with anti-oxidative andanti-inflammatory activity comprising a therapeutically effective amountof the composition of claim 1, wherein said pharmaceutical or dietarysupplement further comprises one or more pharmaceutically acceptableexcipients.
 3. A composition comprising tetrahydroxycurcumin in therange of 30-80%; demethylcurcumin in the range of 4-20%;demethylmonodemethoxy curcumin in the range of 5-25%; andbisdemethoxycurcumin in the range of 0.1-10%.
 4. A pharmaceutical ordietary supplement with anti-oxidative and anti-inflammatory activitycomprising a therapeutically effective amount of the composition ofclaim 3, wherein said pharmaceutical or dietary supplement furthercomprises one or more pharmaceutically acceptable excipients.
 5. Amethod for treating inflammation comprising administering to a mammal inneed thereof, a therapeutically effective amount of the composition ofclaim
 1. 6. The method of claim 5 wherein said mammal is a human.
 7. Amethod for treating inflammation comprising administering to a mammal inneed thereof, a therapeutically effective amount of the composition ofclaim
 3. 8. The method of claim 7 wherein said mammal is a human.